The maturation of the field of microfluidics, the ubiquity of smart camera phones, and the expiration of patents that suppressed novel "lateral flow" rapid diagnostic tests (RDTs), have conspired to produce a renaissance in interest in low-cost diagnostic tests based on paper. Our group has proposed the use of 2-dimensional paper networks (2DPNs) as an extremely versatile approach to rapid immunoassays that will bring the sensitivity and specificity of laboratory-based tests to remote and resource-limited settings. The aim of this project is to apply the instrument-free 2DPN platform to detection of viral antigens;these devices will greatly increase sensitivity, while maintaining the speed, ease of use, and low cost of RDTs. There is a compelling need for a rapid and sensitive point-of-care (POC) diagnostic for influenza (NIAID Category C pathogen) in the U.S. and abroad. Many tests for influenza diagnosis and strain identification have been developed, but all tests in an RDT format have unacceptably low sensitivity;in 2009 the Centers for Disease Control issued a recommendation to discontinue domestic use of currently-available RDTs due to their poor sensitivity. To enhance the sensitivity and reproducibility of the 2DPN-based influenza test proposed here, we will utilize chemical signal amplification, novel high-performance low-cost binding molecules for subtyping influenza strains, and paper substrates with more uniform properties. These innovations will enable us to create a rapid POC diagnostic for influenza that surpasses any currently available rapid flu diagnostics in sensitivity. The final version of the 2DPN influenza test will also have a specificity that is at least comparable to those existing tests. It will require less than 20 minutes from sample to result, be easy to use and interpret by untrained operators with on-board verification of proper test operation, and have a low cost comparable to conventional RDTs. The test will not require lab facilities, equipment or electricity, so will be appropriate for use in remote or resource- limited settings. It will have a multiplexed format for the detection of multiple targets (including flu A, flu B, and subtypes H1 and H3 of flu A) and will be adaptable to the integration of new targets using conventional or de novo reagents developed in the proposed project. PUBLIC HEALTH RELEVANCE: The aim of this project is to develop a novel paper-based diagnostic for the detection of influenza A and B. The device will greatly increase sensitivity, while maintaining the speed, ease of use, and low cost of conventional rapid diagnostic tests, and thus fill a pressing need for point-of-care flu testing in resource-limited settings.